Andrea Feucht

Cytological and biochemical characterization of the FtsA cell division protein of Bacillus subtilis

The actin‐like protein FtsA is present in many eubacteria, and genetic experiments have shown that it plays an important, sometimes essential, role in cell division. Here, we show that Bacillus subtilis FtsA is targeted to division sites in both vegetative and sporulating cells. As in other organisms FtsA is probably recruited immediately after FtsZ. In sporulating cells of B. subtilis FtsZ is recruited to potential division sites at both poles of the cell, but asymmetric division occurs at only one pole. We have now found that FtsA is recruited to only one cell pole, suggesting that it may play an important role in the generation of asymmetry in this system. FtsA is present in much higher quantities in B. subtilis than in Escherichia coli, with approximately one molecule of FtsA for five of FtsZ. This means that there is sufficient FtsA to form a complete circumferential ring at the division site. Therefore, FtsA may have a direct structural role in cell division. We have purified FtsA and shown that it behaves as a dimer and that it has both ATP‐binding and ATP‐hydrolysis activities. This suggests that ATP hydrolysis by FtsA is required, together with GTP hydrolysis by FtsZ, for cell division in B. subtilis (and possibly in most eubacteria).

Direct interaction between the cell division protein FtsZ and the cell differentiation protein SpoIIE.

SpoIIE is a bifunctional protein with two critical roles in the establishment of cell fate in Bacillus subtilis. First, SpoIIE is needed for the normal formation of the asymmetrically positioned septum that forms early in sporulation and separates the mother cell from the prespore compartment. Secondly, SpoIIE is essential for the activation of the first compartment‐specific transcription factor σF in the prespore. After initiation of sporulation, SpoIIE localizes to the potential asymmetric cell division sites near one or both cell poles. Localization of SpoIIE was shown to be dependent on the essential cell division protein FtsZ. To understand how SpoIIE is targeted to the asymmetric septum we have now analysed its interaction with FtsZ in vitro. Using the yeast two‐hybrid system and purified FtsZ, and full‐length and truncated SpoIIE proteins, we demonstrate that the two proteins interact directly and that domain II and possibly domain I of SpoIIE are required for the interaction. Moreover, we show that SpoIIE interacts with itself and suggest that this self‐interaction plays a role in assembly of SpoIIE into the division machinery.

Improved plasmid vectors for the production of multiple fluorescent protein fusions in Bacillus subtilis.

The intrinsically fluorescent green fluorescent protein has been used in many laboratories as a cytological marker to monitor protein localisation in live cells. Multiple spectrally modified mutant versions and novel fluorescent proteins from other species have subsequently been reported and used for labelling cells with multiple fluorescent protein fusions. In this work we report the design and use of vectors containing some of these spectral variants of GFP for use in the Gram positive bacterium Bacillus subtilis. These vectors complement those previously described (Lewis and Marston, 1999. Gene 227, 101-109) to provide a large suite of plasmid vectors for use in this and other related Gram positive organisms. Using these vectors we have been able to directly demonstrate the sequential assembly/disassembly of proteins involved in the generation of cellular asymmetry during development.

CHARACTERIZATION OF A MORPHOLOGICAL CHECKPOINT COUPLING CELL-SPECIFIC TRANSCRIPTION TO SEPTATION IN BACILLUS SUBTILIS

Early in the process of spore formation in Bacillus subtilis, asymmetric cell division produces a large mother cell and a much smaller prespore. Differentiation of the prespore is initiated by activation of an RNA polymerase sigma factor, σF, specifically in that cell. σF is controlled by a regulatory cascade involving an anti‐sigma factor, SpoIIAB, an anti‐anti‐sigma factor, SpoIIAA, and a membrane‐bound phosphatase, SpoIIE, which converts the inactive, phosphorylated form of SpoIIAA back to the active form. SpoIIE is required for proper asymmetric division and much of the protein is sequestered into the prespore during septation. Importantly, activation of σF is dependent on formation of the asymmetric septum. We have now characterized this morphological checkpoint in detail, using strains affected in cell division and/or spoIIE function. Surprisingly, we found that significant dephosphorylation of SpoIIAA occurred even in the absence of septation. This shows that the SpoIIE phosphatase is at least partially active independent of the morphological event and also that cells can tolerate significant levels of unphosphorylated SpoIIAA without activating σF. We also describe a spoIIE mutant in which the checkpoint is bypassed, probably by an increase in the dephosphorylation of SpoIIAA. Taken together, the results support the idea that sequestration of SpoIIE protein into the prespore plays an important role in the control of σF activation and in coupling this activation to septation.

The cell differentiation protein SpoIIE contains a regulatory site that controls its phosphatase activity in response to asymmetric septation.

Summary Starvation induces Bacillus subtilis to initiate a simple, two‐cell developmental process that begins with an asymmetric cell division. Activation of the first compartment‐specific transcription factor, σF, is coupled to this morphological event. SpoIIE, a bifunctional protein, is essential for the compartment‐specific activation of σF and also has a morphogenic activity required for asymmetric cell division. SpoIIE consists of three domains: a hydrophobic N‐terminal domain, which targets the protein to the membrane; a central domain, involved in oligomerization of SpoIIE and interaction with the cell division protein FtsZ; and a C‐terminal domain comprising a PP2C protein phosphatase. Here, we report the isolation of mutations at the very beginning of the central domain of spoIIE, which are capable of activating σF independently of septum formation. Purified mutant proteins showed the same phosphatase activity as the wild‐type protein in vitro. The mutant proteins were fully functional in respect of their localization to sites of asymmetric septation and support of asymmetric division. The data provide strong evidence that the phosphatase domain of SpoIIE is tightly regulated in a way that makes it respond to the formation of the asymmetric septum.

Analysis of the Interaction between the Transcription Factor σG and the Anti-Sigma Factor SpoIIAB of Bacillus subtilis

The activation of sigma(G), a transcription factor, in Bacillus subtilis is coupled to the completion of engulfment during sporulation. SpoIIAB, an anti-sigma factor involved in regulation of sigma(F), is also shown to form a complex with sigma(G) in vitro. SpoIIAA, the corresponding anti-anti-sigma factor, can disrupt the SpoIIAB:sigma(G) complex, releasing free sigma(G). The data suggest the existence of an as-yet-unknown mechanism to keep sigma(G) inactive prior to engulfment.

Regulation of Prespore-Specific Transcription during Sporulation in Bacillus subtilis

During spore formation in the Gram positive bacterium, Bacillus subtilis, asymmetric cell division produces a small prespore cell and a much larger mother cell. The two cells then collaborate in an intricate developmental process which culminates with lysis of the mother cell and release of the mature spore (Fig. 1). Many genes involved in sporulation are known and the regulatory pathways controlling their expression are well understood (Errington, 1993; Stragier and Losick, 1996). The main changes in gene expression during sporulation are controlled by four sigma factors, each of which directs RNA polymerise to recognise new promoter sequences and thus turn on new sets of genes. Two of the sporulation-specific sigma factors act successively in the prespore, σF and then σG: the others act successively in the mother cell; σE followed by σK (Fig. 1). All four sigma factors are tightly regulated, at both transcriptional and post-translational levels. The complex regulation serves to ensure that the sigma factors only become active at the proper time and place in the developmental process. Although the different regulatory programmes of the prespore and mother cell operate in separate compartments, they are by no means independent. Indeed, in genetic experiments the four sigma factor activities behave as if they operate in a linear dependent sequence, thus:

Prespore-specific gene expression in Bacillus subtilis is driven by sequestration of SpoIIE phosphatase to the prespore side of the asymmetric septum

{\sigma ^F} \to {\sigma ^E} \to {\sigma ^G} \to {\sigma ^K}